Bioengineered and emerging pathogens represent a significant threat to human health. The best defense against a rapidly-expanding pandemic is to have capabilities to isolate the causative pathogen quickly from biological samples such that it can be characterized and so that tests and vaccines can be developed against it. Whether the scenario for biological analysis involves samples from the environment, food, water, agriculture, animals, or from humans, the one persistent technology gap in the process of identifying and quantifying the presence of pathogenic agents has been “the front end of assays,” namely sample handling and sample preparation.
One problem encountered in sample handling is separating and concentrating small particles from complex liquid samples. This problem is of particular importance in the applications of pathogen detection and medical diagnostics, wherein separating a particle type of interest (e.g., cells, viruses, bacteria, etc.) from an obscuring background of other materials can increase the sensitivity of a diagnostic assay, and allow particles present at very low concentrations to be detected more easily. Some prior approaches have been based on passive separations relying upon differences in diffusion speeds of different particles or the ability of different particles to negotiate an array of small obstacles or openings. Simple physical filters can be included in this category.
Other approaches have used centrifugal motion to manipulate particles and drive them to separate locations, which has its advantages and disadvantages. The basic slowness and awkwardness of centrifugation is a primary problem with this sample preparation technique, but also its incompatibility with automation or high-throughput parallel processing causes this technique to suffer in the application of rapid virus and biothreat detection systems. It relies on bulky equipment and requires manual manipulation by a technician. It can also be dangerous as the high rotational speeds developed within ultracentrifuges can result in serious accidents if the equipment fails, potentially spreading aerosolized virus over a large area. Many laboratories that work with pathogenic viruses prohibit or limit the use of centrifuges for this reason.
Most standard laboratory methods for viral separation from oral-cavity samples consist of batch procedures based on centrifugation or week-long propagation of viruses. Three critical drawbacks to these techniques are: 1) clinical labs avoid ultracentrifugation of pathogenic samples due to the possibility of aerosolization of the sample (especially following potential equipment failure, as previously stated); 2) all the viruses are coalesced and further processing is required to isolate the pathogen; and 3) these techniques are not amenable to quick, high-throughput processing, which may be necessary to correctly identify the pathogen in a timely fashion.
Separation and purification of viruses from clinical samples that consist of free floating nucleic acids, viruses, bacteria, cells, debris, and/or mucus in isotonic solutions is of particular interest in the identification of potential bio-threats. The approaches to identify potential bio-threats include acoustic focusing of large (>2 um.) particulates, electrophoresis of charged biological particles and molecules, and dielectrophoresis of bacteria. However, none of these can selectively exclude exogenous free-floating nucleic acids, which might hinder amplification and identification of nucleic acids of the target viruses. Adding DNAse or RNAse in the buffer is not effective, as it inhibits PCR process and currently cannot be inactivated in a reliable manner.
The state-of-art nucleic acids purification methods mainly rely on mechanical and/or affinity-based filtration. These conventional methods require long processing times (˜20 min) and relatively expensive reagents (e.g., magnetic microbeads and antibodies), are traditionally difficult to implement in microchip systems, and/or using the Boom capture chemistry, the conventional methods require reagents that may possibly lyse viruses and expose the viruses' internal DNA to the DNA extraction process.
As is true in any such biodetection process, sample preparation is a critical requirement for many biological assays and is a major bottleneck in the process of detecting and identifying biological agents. Capabilities for separation, detection, and classification of unknown species from biological samples becomes more urgent when dealing with bioengineered threats because the investigator must rapidly isolate the unknown from all the other particles in the sample to enable characterization and the development of antibody or nucleic acid-based detection assays. Viruses are an important category of pathogens because some of its members, such as influenza and smallpox, are extremely infectious and very virulent forms could result in sudden, massive pandemics. Viruses are often difficult to isolate due to their small size (typically <200 nm.), compared with the bulk of the particles in a sample. Therefore, since standard laboratory methods cannot rapidly and efficiently separate or purify virus and bacteria from samples, there is an unaddressed need of national importance in rapid isolation, detection, and classification of engineered and naturally-occurring emerging bio-threats.